Role of Receptors in the Metanephric Mesenchyme

描述(申请人提供)：先天性肾脏疾病是儿童慢性肾脏疾病的主要原因。该应用程序的广泛长期目标是了解肾脏发育的分子控制，以对先天性肾脏疾病产生影响。成纤维细胞生长因子受体(FGFRs)通过成纤维细胞生长因子受体底物2(Frs2)等适配蛋白传递信号，对肾脏发育至关重要；然而，由于突变胚胎的早期致死性，全球小鼠基因靶向尚未揭示FGFR或Frs2信号在胎儿肾脏中的全部影响。此外，有条件地删除 后肾间充质(MM)中FGFR1和FGFR2呈Pax3cre线，导致肾发育不全。为了阐明FGFR/Frs2β信号在肾发育后期MM中的作用，我们产生了复合突变体：1.在多发性骨髓瘤发育的后期，FGFR1的缺失；2.FGFR2中Frs2结合位点的全局点突变。FGFR2中Pax3cre基因缺失的突变小鼠(PFLR小鼠)，其FGFR1和FGFR2基因Pax3cre缺失导致的肾脏发育不全逃脱；但是，PFLR小鼠出现输尿管芽(UB)分支缺陷，肾祖细胞(NP)过早耗竭，以及输尿管和肾系的进行性膀胱发生(表明Frs2定向信号在FGFR2下游的重要性)。PFLR小鼠的输尿管缺陷始于扩张和过度增殖的UB末端，似乎与不适当的Ret信号有关。这些小鼠在纤毛结构、典型的Wnt信号和平面细胞极性(PCP)方面也似乎存在缺陷，每种缺陷都可能导致发育和囊性缺陷。另一组由Six2cre系(“sflr”)产生的小鼠，其缺失FGFR1的时间略晚于PFLR，并且仅在NPs(比PLFR更受限)中产生，导致NP耗竭和肾单位血统的包囊，但没有明显的输尿管缺陷。Sflr NPs最初正常形成，但似乎失去了干细胞样标记并经历了凋亡。第三组小鼠由Wnt4cre系(WFLR)产生，其在肾小泡中的缺失晚于sflr，没有明显的肾脏异常。假设后肾间充质中FGFR信号通过Frs2的时空调控对于发育过程中的输尿管和肾源性谱系模式都是至关重要的。为了验证这一假说，产生了以下目的：目的1.表征突变小鼠等位基因系列中的非自主输尿管和自主肾源性谱系缺陷。将对胚胎和出生后的肾脏进行结构和功能缺陷评估。目的2.探讨肾间充质中FGFR/Frs2信号通路导致输尿管形态发生缺陷和膀胱发生的机制。将评估Bmp4/Ret/Erk轴以及纤毛、典型Wnt和PCP缺陷的作用。目的：探讨肾间充质中FGFR/Frs2信号通路导致肾小球祖细胞过早耗竭的机制。通过无偏分析确定的FGFR/Frs2信号的候选靶点将在NP细胞培养中进行评估。

DESCRIPTION (provided by applicant): Congenital kidney diseases are leading causes of chronic kidney disease in children. The application's broad long-term objectives are to understand molecular control of kidney development to make an impact on congenital kidney disease. Fibroblast growth factor receptors (Fgfrs) that signal through adapter proteins such as fibroblast growth factor receptor substrate 2¿ (Frs2¿) are critical for kidney development; however, global mouse gene targeting has not revealed the full impact of Fgfr or Frs2¿ signaling in fetal kidneys due to early lethality of the mutant embryos. Furthermore, conditional deletion of Fgfr1 and Fgfr2 in the metanephric mesenchyme (MM) with a Pax3cre line led to renal dysgenesis. To elucidate roles of Fgfr/Frs2¿ signaling in the MM at later stages of renal development, compound mutants were generated with 1. Deletion of Fgfr1 in increasingly restricted and later stages of MM development and 2. Global point mutations in the Frs2¿ binding site in Fgfr2. The mutant mice with Fgfr1 deletion in early MM with the Pax3cre line and point mutations in the Frs2¿ binding site in Fgfr2 (PFLR mice), escape the renal dysgenesis seen with Pax3cre deletion of Fgfr1 and Fgfr2; however, PFLR mice developed ureteric bud (UB) branching defects, premature nephron progenitor (NP) depletion, and progressive cystogenesis in both ureteric and nephron lineages (showing importance of Frs2¿ directed signaling downstream of Fgfr2). The ureteric defects in PFLR mice start with dilated and hyper- proliferative UB tips that appear to be linked to inappropriate Ret signaling. The mice also appear to have defects in ciliary structure, canonical Wnt signaling, and planar cell polarity (PCP) that each may contribute to the developmental and cystic defects. Another set of mice, generated with a Six2cre line ("SFLR") that deletes Fgfr1 slightly later than PFLR and only in NPs (more restricted than PLFR) cause NP depletion and cysts from the nephron lineages, but have no apparent ureteric defects. SFLR NPs initially form normally, but appear to lose stem cell-like markers and undergo apoptosis. The third set of mice, generated with a Wnt4cre line ("WFLR") that delete in renal vesicles later than SFLR have no apparent renal abnormalities. The hypothesis is that spatial and temporal regulation of Fgfr signaling through Frs2¿ in the metanephric mesenchyme is critical for both ureteric and nephrogenic lineage patterning during development. To test the hypothesis, the following Aims were generated: Aim 1. To characterize the non-autonomous ureteric and autonomous nephrogenic lineage patterning defects in the allelic series of mutant mice. Embryonic and post-natal kidneys will be assessed for structural and functional defects. Aim 2. To determine mechanisms by which Fgfr/Frs2¿ signaling in kidney mesenchyme leads to ureteric morphogenesis defects and cystogenesis. Roles of the Bmp4/Ret/Erk axis and ciliary, canonical Wnt and PCP defects will be assessed. Aim 3: To determine mechanisms by which Fgfr/Frs2¿ signaling in kidney mesenchyme leads to premature nephron progenitor depletion. Candidate targets of Fgfr/Frs2¿ signaling identified by unbiased assays will assessed in NP cell cultures.